RU2804433C2 - Set of str-markers of the y-chromosome for determining the ethno-territorial origin of an individual based on a sample of his dna - Google Patents

Abstract

FIELD: biotechnology; molecular genetics.

SUBSTANCE: invention can be used to identify genotypes to determine a person and the ethnicity of a person based on a sample of his DNA using a system of Y-chromosome microsatellite DNA markers. A set of genetic markers is presented. The set contains synthetic oligonucleotide primers representing the nucleotide sequences of the following SEQ ID NO: 1–70 and defining 38 short tandem repeats localized on the non-recombining region of the Y chromosome (DYS19, DYS385a, DYS385b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS442, DYS445, DYS448, DYS449, DYS456, DYS458, DYS460, DYS481, DYS504, DYS505, DYS518, DYS525, DYS533, DYS537, DYS552, DYS570, DYS576, DYS635, DYS 643, YCAIIa, YCAIIb, GATA-H4.1, Y-GATA-A10, GGAAT1B07, DYF387a, DYF387b). A method of identifying genotypes to determine a person in the Russian population using the above primers is proposed.

EFFECT: selected microsatellite markers are highly informative for most population samples.

2 cl, 2 dwg

Description

The invention relates to the field of molecular genetics and can be used to identify genotypes for personal identification and determination of a person’s ethnicity based on his DNA sample using a system of microsatellite DNA markers of the Y chromosome.

DNA typing is the most conclusive method of analyzing biological material in forensic identification examinations. Molecular genetic identification analysis allows you to examine special sections of DNA that are strictly specific to each individual, and thus obtain a unique genetic “passport” of a person that cannot be hidden, changed or faked. Individualizing characteristics, determined at the DNA level, are characterized by almost absolute stability, that is, they remain unchanged in the human body throughout his entire life.

Over the last decade, for the purposes of genetic identification of an individual, the study of various marker systems localized on autosomes, the non-recombining region of the Y chromosome and in the mitochondrial genome has been widely used. The advantage of non-recombining Y-chromosome lines is that these, having a sufficiently high information content for “general” DNA identification, are transmitted strictly from father to son, which makes it possible to directly establish kinship [1, 2].

Among the world's priority areas in the field of human genetics and medical genetics, the development of test systems aimed at creating a high-tech service for determining the genetic origin of an individual in the male line is currently at the forefront; development of new or improvement of existing genetic test systems used in forensic medicine for personal identification and paternity determination [3, 4]

Polymorphism, used as the main genetic marking system used in forensic practice, results from changes in the number of identical DNA repeats (STRs) within a single locus. The advantage of STR for personal identification and biological relatedness assessment is that it can perform successful analysis even when using highly degraded DNA samples. Advances in genotyping technologies make it possible to easily automate genetic analysis of microsatellites.

DNA identification of a person is based on an analysis of the frequency of occurrence of markers of autosomal loci, the Y chromosome and mtDNA [5, 6]. Analysis of the Y chromosome and (or) mtDNA is necessary in cases where it is necessary to establish paternal or maternal relationships; used when working with highly degraded DNA samples due to the very high copy number of mtDNA; and can also be an alternative to DNA identification by autosomal loci [7] The key point in DNA identification is probabilistic calculations of genotype matches based on reference allele frequencies in the population from which the DNA profile under study comes.

The advantage of STR for personal identification and biological relatedness assessment is that it can perform successful analysis even when using highly degraded DNA samples. Previously, the forensic standard was the “minimal haplotype” of 7 YSTRs, which was later replaced by the “extended haplotype” of 9 loci. To date, YSTR sets have been created that are used for various purposes, including for personal identification.

Currently, the following sets of Y-chromosome DNA markers have been developed and are being produced, which are used for DNA identification:

AmpFlSTR® Yfiler Kit (Applied Biosystems), which allows amplification of 17 STR loci of the Y chromosome in one PCR reaction, which consists of a PCR reaction mixture, a mixture of primers, DNA polymerase, control DNA and an allelic ladder.

PowerPlex®Y23 System (from Promega) for the study of short tandem repeats, allowing the amplification of 23 STR loci of the Y chromosome in one PCR reaction, which consists of a PCR reaction mixture, a mixture of primers, DNA polymerase, control DNA, allelic ladder and deionized water.

Investigator Argus Y-12 QS Kit (Qiagen) for PCR amplification for the study of short tandem repeats, allowing amplification of 12 STR loci of the Y chromosome in one PCR reaction, which consists of a PCR reaction mixture, a mixture of primers, DNA polymerase , control DNA and allelic ladder.

Y-FilerPlus (Applied Biosystems) for the study of short tandem repeats, allowing amplification of 25 STR loci in one PCR reaction.

The currently available developments of sets of Y-chromosome STR markers do not take into account the specifics of the population structure of the Russian population (a large number of ethnic groups, polymorphisms specific to various ethnic groups, not taken into account in foreign developments). For the successful use of Y-STR in criminology and forensic medicine, it is necessary to develop genotyping methods and Y-STR kits, which will maximize the coverage of genetic diversity at these loci in Russian populations. In addition, a significant drawback of existing kits is their significant high cost. Due to the need to use special, expensive consumables (specific DNA polymerases and allelic ladders), the possibilities for widespread use of existing kits in diagnostic laboratories are significantly limited. To eliminate these shortcomings, a necessary condition is to increase the information content of the proposed set and its efficiency.

The register of inventions of the Russian Federation contains 3 similar patents (RU 2558231, RU 2528742, RU 2740575), while only one of the three is valid (RU 2740575). The closest analogues of the set of Y-chromosome STR markers we patent are RU 2528742 (Synthetic oligonucleotide primers and a method for identifying genotypes for personal identification using a system of Y-chromosome microsatellite DNA markers) and the Y-FilerPlus set.

The set of Y-chromosome STR markers we have developed allows us to determine the population affiliation of a male individual by the specificity of his YSTR haplotype. As a result of the specificity of inheritance and the absence of recombination of the Y chromosome, the population specificity of various lines (haplotypes and subhaplogroups), with a selected number of STR markers used, allows for a fairly accurate determination of the ethno-territorial origin of a male individual (paternal line). The selected microsatellite markers are quite highly informative for most population samples. The use of this set of YSTR markers made it possible to move to a more accurate new level of detail and differentiation of populations. The level of information content of haplotypes for selected markers allows one to achieve values that practically allow one to identify differences between population groups. As a result of the analysis of the obtained genotypic data, it was shown that the haplotypes constructed using genotyping of 38 STR markers of the Y chromosome have significant population and territorial specificity. A very high interpopulation differentiation of the obtained YSTR haplotypes has been established, which makes it possible to effectively use the established set of YSTR markers for DNA identification purposes.

A new technical task is to increase the sensitivity, accuracy and information content of the method and create a system of genetic markers covering genetic diversity at 38 loci in Russian populations.

To solve this problem, synthetic oligonucleotide primers (SNP ID NO 1-70) are proposed, complementary to the corresponding sections of fifteen microsatellite loci of the Y chromosome, and pairs of primers are used, one of which has a fluorescent label, and the second is unlabeled, while direct primers for each of the loci carries a fluorescent tag at the 5' end: TET (4, 7, 2', 7'-tetrachloro-6-carboxyfluorescein) for DYS385, DYS390, DYS391, DYS426, DYS436 and DYS439; FAM (6-carboxyfluorescein) for DYS392, DYS393, DYS437 and DYS438; and HEX (4, 7, 2', 4',5',7'-hexachloro-6-carboxyfluorescein) for DYS394, DYS389I, DYS389II DYS388 and DYS434, also synthetic oligonucleotide primers have the following nucleotide composition:

DYS385 (SNP ID NO: 1) F: 5'-TET-AGCATGGGTGACAGAGCTA-3'

DYS385 (SNP ID NO: 2) R: 5'-TGGGATGCTAGGTAAAGCTG

DYS389 (SNP ID NO: 3) F: 5'-HEX-CCAACTCTCATCTGTATTATCTATG

DYS389 (SNP ID NO: 4) R: 5'-TCTTATCTCCACCCACCAGA

DYS390 (SNP ID NO: 5) F: 5'-TET-TATATTTTACACATTTTTGGGCC

DYS390 (SNP ID NO: 6) R: 5'-TGACAGTAAAATGAACACATTGC

DYS391 (SNP ID NO: 7) F: 5'-TET-CTATTCATTCAATCATACACCCA

DYS391 (SNP ID NO: 8) R: GATTCTTTGTGGTGGGTCTG

DYS392 (SNP ID NO: 9) F: 5'-FAM-TCATTAATCTAGCTTTTAAAAACAA

DYS392 (SNP ID NO: 10) R: 5'-AGACCCAGTTGATGCAATGT

DYS393 (SNP ID NO: 11) F: 5'-FAM-GTGGTCTTCTACTTGTGTCAATAC

DYS393 (SNP ID NO: 12) R: 5'-AACTCAAGTCCAAAAATGAGG

DYS394 (SNP ID NO: 13) F: 5'-HEX-CTACTGAGTTTCTGTTATAGT

DYS394 (SNP ID NO: 14) R: 5'-ATGGCATGTAGTGAGGACA

DYS437 (SNP ID NO: 15) F: 5'-FAM-GACTATGGGCGTGAGTGCAT-3'

DYS437 (SNP ID NO: 16) R: 5'-GAGACCCTGTCATTCACAGATGA

DYS438 (SNP ID NO: 17) F: 5'-FAM-CCAAAATTAGTGGGGAATAGTTG-3'

DYS438 (SNP ID NO: 18) R: 5'-GATCACCCAGGGTCTGGAGTT-3'

DYS439 (SNP ID NO: 19) F: 5'-TET-TCGAGTTGTTATGGTTTTAGGTCT-3'

DYS439 (SNP ID NO: 20) R: 5'-GTGGCTTGGAATTCTTTTACCC-3'

DYS442 (SNP ID NO: 21) F: 5'-HEX-CCCCAAGTCCCCAAAGTGTGT-3'

DYS442 (SNP ID NO: 22) R: 5'-CACTCATTGATTGATGGGCGTTT-3'

DYS445 (SNP ID NO: 23) F: 5'-FAM-AGTTAAGAGCCCCACCTTCCTG-3'

DYS445 (SNP ID NO: 24) R: 5'-TTTTGGTGGCATAATCTCAGCTC-3'

DYS448 (SNP ID NO: 25) F: 5'-FAM-TGGGAGAGGCAAGGATCCAA-3'

DYS448 (SNP ID NO: 26) R: 5'-CCAGACCGGCCAGAAATATGAC-3'

DYS449 (SNP ID NO: 27) F: 5'-FAM-TTTTCCCTTAACTTGTGTGATTTTT-3'

DYS449 (SNP ID NO: 28) R: 5'-AGGTTGGACAACAAGAGTAAGACAG-3'

DYS456 (SNP ID NO: 29) F: 5'-FAM-GGACCTTGTGATAATGTA-3'

DYS456 (SNP ID NO: 30) R: 5'-GTTTTGGGCTGAGTTGATGGG-3'

DYS458 (SNP ID NO: 31) F: 5'-FAM-AGCAACAGGAATGAAACTCCAAT-3'

DYS458 (SNP ID NO: 32) R: 5'-GGAGGGTGGGCGTGGTGG-3'

DYS460 (SNP ID NO: 33) F: 5'-ROX-GAGGAATCTGACACCTCTGACA-3'

DYS460 (SNP ID NO: 34) R: 5'-GTCCATATCATCTATCCTCTGCCTA-3'

DYS481 (SNP ID NO: 35) F: 5'-JOE-AGGAATGTGGCTAACGCTGT-3'

DYS481 (SNP ID NO: 36) R: 5'-ACAGCTCACCAGAAGGTTGC-3'

DYS504 (SNP ID NO: 37) F: 5'-HEX-TCTACACCACTGTGCCAAGC-3'

DYS504 (SNP ID NO: 38) R: 5'-GGCAACAGAGCAACCCCTCT-3'

DYS505 (SNP ID NO: 39) F: 5'-FAM-TCTGGCGAAGTAACCCAAAC-3'

DYS505 (SNP ID NO: 40) R: 5'-TCGAGTCAGTTCACCAGAAGG-3'

DYS518 (SNP ID NO: 41) F: 5'-JOE-GGCAACACAAGTGAAACTGC-3'

DYS518 (SNP ID NO: 42) R: 5'-TCAGCTCTTACCATGGGTGAT-3'

DYS525 (SNP ID NO: 43) F: 5'-HEX-ATTCACACCATTGCACTCCA-3'

DYS525 (SNP ID NO: 44) R: 5'-GAGGCGGAGCTTTTAGTGAG-3'

DYS531 (SNP ID NO: 45) F: 5'-FAM-GACCCACTGGCATTCAAATC-3'

DYS531 (SNP ID NO: 46) R: 5'-TGCTCCCTTTCTTTGTAGACG-3'

DYS533 (SNP ID NO: 47) F: 5'-JOE-CATCTAACATCTTTGTCATCTACC-3'

DYS533 (SNP ID NO: 48) R: 5'-TGATCAGTTCTTAACTCAACCAA-3'

DYS537 (SNP ID NO: 49) F: 5'-HEX-GGTCTCCAATTCCATCCAGA-3'

DYS537 (SNP ID NO: 50) R: 5'-TGGAACATGCCCATTAATCA-3'

DYS552 (SNP ID NO: 51) F: 5'-FAM-CCATAGTGCCGAGGTCAAGT-3'

DYS552 (SNP ID NO: 52) R: 5'-AACACCTG ATGCCTGGTTG-3'

DYS570 (SNP ID NO: 53) F: 5'-ROX-GAACTGTCTACAATGGCTCACG-3'

DYS570 (SNP ID NO: 54) R: 5'-TCAGCATAGTCAAGAAACCAGACAAC-3'

DYS576 (SNP ID NO: 55) F: 5'-ROX-TGGGCTGAGGAGTTCAATC-3'

DYS576 (SNP ID NO: 56) R: 5'-GGCAGTCTCATTTCCTGGAGATG

DYS635 (SNP ID NO: 57) F: 5'-FAM-AGTGTCTCACTTCAAGCACCAAGCAC-3'

DYS635 (SNP ID NO: 58) R: 5'-ACATTTTTTCCAACTGTGAATTTTGCTGC-3'

DYS643 (SNP ID NO: 59) F: 5'-FAM-AAGCCATGCCTGGTTAAACT-3'

DYS643 (SNP ID NO: 60) R: 5'-TGTAACCAAACACCACCCATT-3'

YCAII (SNP ID NO: 61) F: 5'-HEX-TGTCAAAATTTAACCCACAATCA-3'

YCAII (SNP ID NO: 62) R: 5'-GCAGTCTTTCACCATAAGGTTAGC-3'

GATA H4.1 (SNP ID NO: 63) F: 5'-HEX-ATGCTGAGGAGAATTTCCAA-3'

GATA H4.1 (SNP ID NO: 64) R: 5'-GCTATTCATCCATCTAATCTATCCATT-3'

Y-GATA-A10 (SNP ID NO: 65)

F: 5'-NED-CCTGCCATCTCTATTTATCTTGCATATA-3'

Y-GATA-A10 (SNP ID NO: 66) R: 5'-ATAAATGGAGATAGTGGGTGGATT-3'

GGAAT1B07 (SNP ID NO: 67) F: 5'-NED-CATTAACCATAGCATTTCTTTCC-3'

GGAAT1B07 (SNP ID NO: 68) R: 5'-GAAGTAGAGTGGAATGGACTGC-3'

DYF387S1 (SNP ID NO: 69) F: 5'-FAM-GCCTGGGTGACAGAGCTAGA-3'

DYF387S1 (SNP ID NO: 70) R: 5'-GCCACAGTGTGAGAAGTGTGA-3'

In the method of identifying genotypes for personal identification using a system of microsatellite DNA markers of the Y chromosome, the above primers are used, while genotyping of 38 short tandem repeats localized on the non-recombining region of the Y chromosome (YSTR) is carried out on a genetic DNA analyzer using a capillary gel electrophoresis, using the PCR method and fragment analysis of fluorescently labeled DNA amplicons, use male DNA samples isolated from any biological material in accordance with standard methods that ensure high-quality purification of DNA from proteins and impurities and prevent degradation of the material during isolation, with carrying out capillary electrophoresis of fragments synthesized during PCR, having a fluorescent TET label (4, 7, 2', 7'-tetrachloro-6-carboxyfluorescein) in the length range 360-420 bp. correspond to alleles 9-23 tandem repeats in the DYS385 locus, fragments having a TET fluorescent tag in the length range 196-224 bp. correspond to alleles of 19-26 tandem repeats in the DYS390 locus, fragments having a TET fluorescent tag in the length range 277-289 bp. correspond to alleles of 9-12 tandem repeats in the DYS391 locus, fragments having a TET fluorescent tag in the length range 232-260 bp. correspond to alleles of 8-15 tandem repeats in the DYS439 locus, fragments having a fluorescent FAM (6-carboxyfluorescein) label in the length range 243-259 bp. correspond to alleles of 10-15 tandem repeats in the DYS392 locus, fragments having a fluorescent FAM tag in the length range of 116-128 bp. correspond to alleles of 12-15 tandem repeats in the DYS393 locus, fragments having a fluorescent FAM tag in the length range of 174-190 bp. correspond to alleles of 6-10 tandem repeats in the DYS437 locus, fragments having a fluorescent FAM tag in the length range 311-326 bp. correspond to alleles of 9-12 tandem repeats in the DYS438 locus, fragments having a fluorescent FAM tag in the length range 311-326 bp. correspond to alleles of 9-12 tandem repeats in the DYS438 locus, fragments having a fluorescent FAM tag in the length range 251-263 bp. correspond to alleles of 9-12 tandem repeats in the DYS445 locus, fragments having a fluorescent FAM tag in the length range 307-343 bp. correspond to alleles of 18-24 tandem repeats in the DYS448 locus, fragments having a fluorescent FAM tag in the length range 264-313 bp. correspond to alleles of 25-37 tandem repeats in the DYS449 locus, fragments having a fluorescent FAM tag in the length range 141-157 bp. correspond to alleles of 17-17 tandem repeats in the DYS456 locus, fragments having a fluorescent FAM tag in the length range 106-156 bp. correspond to alleles of 12-24 tandem repeats in the DYS458 locus, fragments having a fluorescent FAM tag in the length range of 165-181 bp. correspond to alleles of 11-15 tandem repeats in the DYS505 locus, fragments having a fluorescent FAM tag in the length range 102-127 bp. correspond to alleles of 8-14 tandem repeats in the DYS531 locus, fragments having a fluorescent FAM tag in the length range of 236-256 bp. correspond to alleles of 7-12 tandem repeats in the DYS552 locus, fragments having a fluorescent FAM tag in the length range 238-274 bp. correspond to alleles of 17-26 tandem repeats in the DYS635 locus, fragments having a fluorescent FAM tag in the length range 123-158 bp. correspond to alleles of 7-14 tandem repeats in the DYS643 locus, fragments having a fluorescent FAM tag in the length range of 240-272 bp. correspond to alleles of 27-35 tandem repeats in the DYF387 locus, fragments having a fluorescent HEX tag (4, 7, 2', 4', 5', 7'-hexachloro-6-carboxyfluorescein) in the length range 190-206 bp. correspond to alleles of 13-17 tandem repeats in the DYS394 locus, fragments having a HEX fluorescent tag in the length range 242-262 bp. correspond to alleles of 7-12 tandem repeats in the DYS389I locus, fragments having a fluorescent HEX tag in the length range 360-382 bp. correspond to the DYS389II locus, the alleles of the DYS389II locus are determined by subtracting the length of the fragment VU83891 from the fragment length value of the range 360-382, the resulting range is 110-134 bp. corresponds to 14-20 tandem repeats, fragments having a fluorescent HEX tag in the length range 298-322 bp. correspond to alleles of 10-16 tandem repeats in the DYS442 locus, fragments having a HEX fluorescent tag in the length range of 265-296 bp. correspond to alleles of 12-20 tandem repeats in the DYS504 locus, fragments having a HEX fluorescent tag in the length range 305-321 bp. correspond to alleles of 9-13 tandem repeats in the DYS525 locus, fragments having a fluorescent HEX tag in the length range 155-179 bp. correspond to alleles of 7-13 tandem repeats in the DYS537 locus, fragments having a fluorescent HEX tag in the length range 147-165 bp. correspond to alleles of 17-26 tandem repeats in the YCAII locus, fragments having a fluorescent HEX tag in the length range 134-152 bp. correspond to alleles of 9-14 tandem repeats in the Y-GATA-H4.1 locus, fragments having a fluorescent ROX (carboxy-X-rhodamine) tag in the length range of 102-122 bp. correspond to alleles of 8-13 tandem repeats in the DYS460 locus, fragments having a fluorescent ROX tag in the length range 242-282 bp. correspond to alleles of 13-23 tandem repeats in the DYS570 locus, fragments having a fluorescent ROX tag in the length range 168-204 bp. correspond to alleles of 13-22 tandem repeats in the DYS576 locus, fragments having a JOE fluorescent label in the length range 117-157 bp. correspond to alleles 189-32 tandem repeats in the DYS481 locus, fragments having a fluorescent label JOE (b-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein) in the length range 252-296 bp. correspond to alleles of 25-36 tandem repeats in the DYS518 locus, fragments having a JOE fluorescent label in the length range of 200-224 bp. correspond to alleles of 9-15 tandem repeats in the DYS533 locus.

The proposed method consists of genotyping 38 STR markers of the Y chromosome on a genetic DNA analyzer using capillary gel electrophoresis, using the PCR method and fragment analysis of fluorescently labeled DNA amplicons. The developed system of genetic markers of the Y chromosome (YSTR) is informative for DNA identification in Russian populations and is based on multiplex genotyping using PCR and capillary gel electrophoresis.

The method is intended:

- to identify genotypes of microsatellite (STR) loci of the human Y chromosome in DNA samples obtained from biological material of males for genetic identification of a person in medical diagnostic, forensic and forensic laboratories;

- to establish a person’s ethnicity based on the structure of his YSTR haplotype;

- to establish family ties, including when investigating cases of disputed paternity;

- for scientific research in the field of human population genetics and medical genetics.

The developed method consists of using specially selected highly specific fluorescently labeled oligonucleotide primers to carry out a polymerase chain reaction (PCR) to amplify microsatellite markers of the human Y chromosome.

The proposed method makes it possible to obtain a complex genotype of the studied human DNA sample using 38 microsatellite markers of the non-recombining part of the Y chromosome: DYS19, DYS385a, DYS385b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DY S439, DYS442, DYS445, DYS448, DYS449, DYS456, DYS458, DYS460, DYS481, DYS504, DYS505, DYS518, DYS525, DYS533, DYS537, DYS552, DYS570, DYS576, DYS635, DYS 643, YCAIIa, YCAIIb , GATA-H4.1, Y-GATA-A10, GGAAT1B07 , DYF387a, DYF387b.

Genotypes are presented as the size of the fragment lengths of the corresponding amplicons in nucleotides. It is this wide set of YSTRs that is very informative for population genetic and forensic studies. An assessment of the information content of the selected markers using available literature data and genetic databases showed the high potential of this set of microsatellite markers of the non-recombining part of the Y chromosome for genetic differentiation of populations and individuals and linking a person to a specific ethnic group based on the specificity of his haplotype within various haplogroups of the Y chromosome.

The method is carried out as follows

For this work, we use DNA samples isolated from any human biological material in accordance with standard methods that ensure high-quality DNA purification from proteins and impurities and prevent degradation of the material during isolation. The concentration of the working solution of total undegraded DNA for PCR must be at least 2 nanograms per microliter.

Genotyping of microsatellite DNA markers is carried out in several stages, including preparation of DNA samples, polymerase chain reaction (PCR), sample preparation of amplification products, capillary gel electrophoresis on automatic DNA analyzers, computer program processing of primary data and, in fact, obtaining the final genotypes of the subjects under study. samples.

Multiplex genotyping includes several sequential steps:

- obtaining PCR products containing sequences with the studied YSTR in the test sample;

- combining all the studied markers in a common mixture of PCR products;

- purification of the amplicon mixture from mineral oil (if PCR was carried out with the addition of mineral oil in amplifiers without a heated lid);

- preparation of a mixture of PCR products with a matrix length standard and formamide;

- denaturation of the reaction product in formamide;

- capillary electrophoresis on an automatic analyzer;

- software processing of primary data;

- obtaining the final genotypes of the studied samples.

To amplify selected YSTR markers, a polymerase chain reaction is performed. To effectively separate all products according to their physical size on capillary gel electrophoresis, primers are selected accordingly. The proposed primers are selected from a variety of options for the most optimal combination of lengths of amplified products, separation of fluorescent labels, effective differentiation of alleles of different loci that are close in size, and combination of dyes with standard sets of reagents for optical calibration of automatic DNA analyzers.

Each of the dyes used has its own wavelength of maximum fluorescence (although the emission spectra overlap). A computer program then analyzes the fluorescence intensity and identifies the peaks of the PCR products based on which wavelength there was a maximum increase in fluorescence compared to background levels. The results are ultimately presented in the form of multi-colored peaks (each color corresponds to a specific fluorescent dye) - electrophore grams, with the size of the products indicated, which is established by the length standard added during sample preparation to each sample.

The primers on the DYS385, DYS389, YCAII and DYF387 loci are selected in such a way that simultaneously amplifying, respectively, two microsatellite repeats DYS385a, DYS385b, DYS389II, which produces a longer fragment (353-385 bp), and DYS389I, to which the fragments correspond 239-263 bp long. (Cooper et al., 1996). The number of repeats in the DYS389II locus is determined by subtracting the length of the smaller fragment from the length of the larger fragment.

PCR is carried out in test tubes or plates with a volume of 0.2 or 0.5 ml. The volume of the reaction mixture can vary from 10 to 50 µl. Recommended volume 15 µl. The reaction mixture for PCR, with a total volume of 15 μl, includes 0.5-1.5 μl of specific primers with a concentration of 1 p.u./ml; 0.2 mM concentration of each of the four dNTPs (typically 1 μl of an 8 mM solution, including 2 mM of each dNTP); from 10 to 100 ng of the test sample of genomic DNA; buffer for Taq polymerase and 0.25-1.0 units. Taq DNA polymerases. The reaction is carried out in specialized thermal cyclers (amplifiers).

The selection of conditions for carrying out a polymerase chain reaction using a set of fluorescently labeled primers included an assessment of the quantity and quality of the yield of amplification products of all fifteen STR loci, the presence of nonspecific amplification, the presence of various aberrations during capillary electrophoresis, differences in the intensity of the height of the detected peaks of the products, the number of primers, not included in the reaction. A comparison was made between different buffers, different concentrations of Mg++ ions, primer mixture concentrations, dNTP concentrations, and the total volume of the PCR mixture. As a result, optimal conditions for carrying out PCR were selected.

It is recommended to use modern thermal cyclers for 96-well thin-walled plates: Applied Biosystems, Forster City, USA; Biometra, Germany; I-Cycler, BioRad, USA. The polymerase chain reaction scheme consists of primary denaturation of the DNA template (4 minutes at 94-95°C), then 40 cycles consisting of denaturation (94°C), primer annealing at a temperature specific for each polymorphism (usually from 58°C to 62°C), chain elongation (at 72°C); the reaction is completed by final chain elongation (5-10 minutes at 72°C).

Program for amplification of loci DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS437, DYS442, DYS445, DYS448, DYS449, DYS481, DYS505, DYS518, DYS525, DYS533 , DYS537, DYS552, GATA-H4.1:

Initial denaturation: 94°C - 4 min.

40 cycles: 94°C - 30 sec

58°C - 30 sec

72°C - 45 sec

Final elongation: 72°C - 4 min.

Program for amplification of loci DYS385a, DYS385b, DYS393, DYS438, DYS439, DYS456, DYS458, DYS460, DYS481, DYS570, DYS576, DYS635, DYS 643, YCAIIa, YCAIIb, Y-GATA-A10 , GGAAT1B07, DYF387a, DYF387b:

Initial denaturation: 94°C - 4 min.

40 cycles: 94°C - 30 sec

60°C - 30 sec

72°C - 45 sec

Final elongation: 72°C - 4 min.

Program for amplification of loci DYS437, DYS438:

Initial denaturation: 94°C - 4 min.

40 cycles: 94°C - 30 sec

62°C - 30 sec

72°C - 45 sec

Final elongation: 72°C - 4 min.

Comparative testing of various Russian-made DNA polymerases recommended by manufacturers for PCR. Four different DNA polymerases were compared. The best results based on all tests were obtained using Hot Start Taq DNA polymerase from SibEnzyme (Novosibirsk).

The resulting PCR products of individual loci are combined in a denaturing buffer. To do this, equal amounts of PCR products of all studied YSTR markers (amlicons) are mixed in separate tubes (or plates). The recommended volume of each marker is 5 µl.

All PCR products are diluted with deionized water to a concentration of 10 pM/μL (10 kmM solution). The initial concentration depends on the quality and concentration of the initial DNA template, PCR optimization, the polymerase used and consumables. The relative proportions of mixed products under the operating conditions of a particular laboratory may require correction by experiment.

The denaturation reaction of PCR products in formamide is carried out in a total volume of 10 µl. 2 μl of a mixture of PCR products, 0.5 μl of a molecular weight marker (Gene-Scan-500 ROX size standard), and 9 μl of Hi-Di formamide are transferred into test tubes or plates for an automatic analyzer. The size standard is a mixture of artificially synthesized oligonucleotide fragments from 35 to 500 base pairs in length (35, 50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490 and 500) with chemically grafted with a fluorescent group in solution, when irradiated, fluoresces in the red spectrum. The resulting mixture is denatured for 5 minutes. at 95°C, then immediately cool, either in ice or at 4°C (5 minutes), and then briefly centrifuge.

Genotyping of microsatellite loci of the Y chromosome is carried out using capillary gel electrophoresis. PCR products of individual loci are combined in denaturing buffer and separated on Applied Biosystems Genetic Analyzers. Automatic DNA analyzers are an automated system of instruments designed to determine the nucleotide sequence, or the size and number of DNA fragments. The genetic analyzer is a capillary electrophoresis system with laser-induced fluorescence. DNA samples labeled with up to 4 different fluorescent dyes are collected and placed into the instrument's autosampler. Electrophoresis of dye-labeled DNA fragments occurs in a polymer in which they are separated according to their size. As they pass through the capillary, the labeled samples pass through a special window in the capillary and are irradiated. Fluorescent dyes bound to DNA fragments are excited using a laser and emit light at different wavelengths characteristic of each dye, separated according to wavelengths using a spectrograph. They are stored in a charge-coupled device (CCD) chamber so that all 4 types of fluorescent emission can be detected simultaneously. Using software, light signals are accumulated in intensity using filters selected by the program (spectral or wavelength filters) and stored as electrical signals for subsequent data processing.

The results are ultimately presented in the form of multi-colored peaks (each color corresponds to a specific fluorescent dye) - an electropherogram, with the size of the products indicated, which is established according to the length standard added during sample preparation to each sample. Since genotyping is carried out only using DNA samples from men, the observed combinations of genotypes of different YSTRs reflect their physical location on the Y chromosome of a particular individual.

Figure 1 shows the separation electropherogram of the DNA length standard (GeneScan500-ROX) - red peaks. Figure 2 shows an electropherogram of the separation of microsatellite markers of the Y chromosome by capillary gel electrophoresis in one of the individuals.

The developed method makes it possible to obtain DNA amplifiers containing at least 90% of the volume of specific DNA fragments. Non-specific DNA fragments are allowed in a volume not exceeding 10% of the volume (estimated as the area under the peak of the corresponding specific DNA fragments). Genotyping accuracy is at least 99%. The error in measuring the length of a DNA fragment is no more than 1 pair of nucleotides. Reproducibility of results must be ensured by strict compliance with all geotyping conditions.

Thus, as a result of using the proposed method for genotyping YSTR markers by performing PCR and fragment analysis of the DNA samples under study, it is possible to establish the complex genotype of the human DNA sample under study using 38 microsatellite markers of the non-recombining part of the Y chromosome: DYS19, DYS385a, DYS385b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS442, DYS445, DYS448, DYS449, DYS456, DYS458, DYS460, DYS481, DYS504, DYS505, DYS 518, DYS525, DYS533, DYS537, DYS552, DYS570, DYS576, DYS635, DYS 643, YCAIIa, YCAIIb, GATA-H4.1, Y-GATA-A10, GGAAT1B07, DYF387a, DYF387b.

The proposed method is characterized by high sensitivity, specificity, high accuracy, has a high discriminatory potential and is more economical.

Brief description of drawings

Fig. 1 - Electropherogram of DNA length standard separation (GeneScan500-ROX) red peaks.

The figure shows an electropherogram of the separation of the length standard for determining the sizes of STR marker products by capillary electrophoresis carried out on a sequencer. The electropherogram contains an image of only the length standard, without the addition of the DNA samples being studied.

Fig. 2 - Electropherogram of separation of Y-chromosome microsatellite markers using capillary gel electrophoresis in one of the individuals.

The figure shows an electropherogram of the separation of PCR products of the studied STR markers included in the set, with various fluorescent labels. The electropherogram includes PCR products of YSTR markers from one sample belonging to a man and a linear length standard to determine the size of the fragments of the markers under study.

Information sources

1. Jobling M.A. and Tyler-Smith S.New uses for new haplotypes the human Y chromosome, disease and selection // Trends in Genetics. 2000. V. 16. P. 356-362.

2. Jobling M.A., Tyler-Smith C. Fathers and sons: the Y chromosome and human evolution // Trends in Genetics. 1995. V. 11. P. 449 456.

3. Carracedo A., Bar W., Lincoln P., Mayr W., Morling N., Olaisen V., Schneider P., Budowle V., Brinkmann V., Gil P., Holland M., Tully G, Wilson M., DNA Commission of the hiternational Society for Forensic Genetics: guidelines for mitochondrial DNA typing // Forensic Sci. Int. 2000. Vol.110. - P. 79-85.

4. Sanchez, JJ, Phillips, C, Borsting, C et al. (2006) A multiplex assay with 52 single nucleotide polymorphisms for human identification. Electrophoresis/ 2006. V. 27, (9). P. 1713-1724.

5. Butler J.M., Schoske R., Vallone P.M. et al. A novel multiplex for simultaneous amplification of 20 Y chromosome STR markers // Forensic Sci Int. 2002. V.129. P. 10-24.

6. Zhivotovsky L.A., Akhmetova V.L., Fedorova S.A., Zhirkova V.V., Khusnutdininova E.K. Developing STR databases on structured populations: the native South Siberian population versus the Russian population.Forensic Sci Int; Genetics. 2009. V.3. P. 111-116.

7. Stepanov B.A., Balanovsky O.P., Melnikov A.B., Lash-Zavada A.Yu., Kharkov V.N., Tyazhelova T.V., Akhmetova V.L., Zhukova O.V., Shneider Yu.V. ., Shilnikova N.N., Borinskaya S.A., Marusin A.V., Spiridonova M.G., Simonova K.V., Khitrinskaya I.Yu., Radzhabov M.O., Romanov A.G., Shtygasheva O.V., Koshel S.M., Balanovskaya E.V., Rybakova A.V., Khusnutdinova E.K., Puzyrev V.P., Yankovsky N.K. Characteristics of the populations of the Russian Federation based on a panel of fifteen loci used for DNA identification and forensic examination // Acta Naturae. 2011. T. 3. No. 2. pp. 59-71.

8. RU 2558231 “Method, test system and primers for determining haplogroups of the human Y chromosome.”

9. RU 2528742 “Synthetic oligonucleotide primers and a method for identifying genotypes for personal identification using a system of microsatellite DNA markers of the Y chromosome.”

10. RU 2740575 “A set of synthetic oligonucleotides for simultaneous genotyping of 63 DNA markers associated with the ABO blood group, the main haplogroups of the Y chromosome, the color of the iris, hair, skin and gender, using the PCR method with subsequent hybridization.”

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LIST OF SEQUENCES

<110> Research Institute of Medical Genetics of the Federal

State budgetary scientific institution Tomsk National

research medical center of the Russian Academy of Sciences (Research

Institute of Medical Genetics, Tomsk NRMC)

Federal State Budgetary Institution of Science Institute of General

genetics named after N.I. Vavilov Institute of Russian Academy of Sciences (Vavilov Institute of

General Genetics Russian Academy of Sciences)

<120> Set of STR markers of the Y chromosome for determining ethno-territorial

origin of an individual based on his DNA

<130>

<160> 70

<210> 1

<211> 19

<212> DNA

<213> Homo sapiens

<400> 1

agcatgggtg acagagcta

<210> 2

<211> 20

<212> DNA

<213> Homo sapiens

<400> 2

tgggatgcta ggtaaagctg

<210> 3

<211> 25

<212> DNA

<213> Homo sapiens

<400> 3 26

ccaactctca tctgtattat ctatg

<210> 4

<211> 20

<212> DNA

<213> Homo sapiens

<400> 4

tcttatctcc acccaccaga

<210> 5

<211> 23

<212> DNA

<213> Homo sapiens

<400> 5

tatattttac acatttttgg gcc

<210> 6

<211> 23

<212> DNA

<213> Homo sapiens

<400> 6

tgacagtaaa atgaacacat tgc

<210> 7

<211> 23

<212> DNA

<213> Homo sapiens

<400> 7

ctattcattc aatcatacac cca

<210> 8

<211> 20 27

<212> DNA

<213> Homo sapiens

<400> 8

gattctttgt ggtgggtctg

<210> 9

<211> 25

<212> DNA

<213> Homo sapiens

<400> 9

tcattaatct agcttttaaa aacaa

<210> 10

<211> 20

<212> DNA

<213> Homo sapiens

<400> 10

agacccagtt gatgcaatgt

<210> 11

<211> 24

<212> DNA

<213> Homo sapiens

<400> 11

gtggtcttct acttgtgtca atac

<210> 12

<211> 22

<212> DNA

<213> Homo sapiens

<400> 12 28

aactcaagtc caaaaaatga gg

<210> 13

<211> 21

<212> DNA

<213> Homo sapiens

<400> 13

ctactgagtt tctgttatag t

<210> 14

<211> 19

<212> DNA

<213> Homo sapiens

<400> 14

atggcatgta gtgaggaca

<210> 15

<211> 20

<212> DNA

<213> Homo sapiens

<400> 15

gactatgggc gtgagtgcat

<210> 16

<211> 23

<212> DNA

<213> Homo sapiens

<400> 16

gagaccctgt cattcacaga tga

<210> 17

<211> 23 29

<212> DNA

<213> Homo sapiens

<400> 17

ccaaaattag tggggaatag ttg

<210> 18

<211> 21

<212> DNA

<213> Homo sapiens

<400> 18

gatcaccag ggtctggagt t

<210> 19

<211> 24

<212> DNA

<213> Homo sapiens

<400> 19

tcgagttgtt atggttttag gtct

<210> 20

<211> 22

<212> DNA

<213> Homo sapiens

<400> 20

gtggcttgga attcttttac cc

<210> 21

<211> 21

<212> DNA

<213> Homo sapiens

<400> 21 30

ccccaagtcc ccaaagtgtg t

<210> 22

<211> 23

<212> DNA

<213> Homo sapiens

<400> 22

cactcattga ttgatgggcg ttt

<210> 23

<211> 22

<212> DNA

<213> Homo sapiens

<400> 23

agttaagagc cccaccttcc tg

<210> 24

<211> 23

<212> DNA

<213> Homo sapiens

<400> 24

ttttggtggc ataatctcag ctc

<210> 25

<211> 20

<212> DNA

<213> Homo sapiens

<400> 25

tggggagaggc aaggatccaa

<210> 26

<211> 22 31

<212> DNA

<213> Homo sapiens

<400> 26

ccagaccggc cagaaatatg ac

<210> 27

<211> 25

<212> DNA

<213> Homo sapiens

<400> 27

ttttccctta acttgtgtga ttttt

<210> 28

<211> 25

<212> DNA

<213> Homo sapiens

<400> 28

aggttggaca acaagagtaa gacag

<210> 29

<211> 18

<212> DNA

<213> Homo sapiens

<400> 29

ggaccttgtg ataatgta

<210> 30

<211> 21

<212> DNA

<213> Homo sapiens

<400> 30 32

gttttgggct gagttgatgg g

<210> 31

<211> 23

<212> DNA

<213> Homo sapiens

<400> 31

agcaacagga atgaaactcc aat

<210> 32

<211> 18

<212> DNA

<213> Homo sapiens

<400> 32

ggagggtggg cgtggtgg

<210> 33

<211> 22

<212> DNA

<213> Homo sapiens

<400> 33

gaggaatctg acacctctga ca

<210> 34

<211> 25

<212> DNA

<213> Homo sapiens

<400> 34

gtccatatca tctatcctct gccta

<210> 35

<211> 20 33

<212> DNA

<213> Homo sapiens

<400> 35

aggaatgtgg ctaacgctgt

<210> 36

<211> 20

<212> DNA

<213> Homo sapiens

<400> 36

acagctcacc agaaggttgc

<210> 37

<211> 20

<212> DNA

<213> Homo sapiens

<400> 37

tctacaccac tgtgccaagc

<210> 38

<211> 19

<212> DNA

<213> Homo sapiens

<400> 38

ggcaacagag caaccctct

<210> 39

<211> 20

<212> DNA

<213> Homo sapiens

<400> 39 34

tctggcgaag taacccaaac

<210> 40

<211> 21

<212> DNA

<213> Homo sapiens

<400> 40

tcgagtcagt tcaccagaag g

<210> 41

<211> 20

<212> DNA

<213> Homo sapiens

<400> 41

ggcaacacaa gtgaaactgc

<210> 42

<211> 21

<212> DNA

<213> Homo sapiens

<400> 42

tcagctctta ccatggggtga t

<210> 43

<211> 20

<212> DNA

<213> Homo sapiens

<400> 43

attcacacca ttgcactcca

<210> 44

<211> 20 35

<212> DNA

<213> Homo sapiens

<400> 44

gaggcggagc ttttagtgag

<210> 45

<211> 20

<212> DNA

<213> Homo sapiens

<400> 45

gacccactgg cattcaaatc

<210> 46

<211> 21

<212> DNA

<213> Homo sapiens

<400> 46

tgctcccttt ctttgtagac g

<210> 47

<211> 24

<212> DNA

<213> Homo sapiens

<400> 47

catctaacat ctttgtcatc tacc

<210> 48

<211> 23

<212> DNA

<213> Homo sapiens

<400> 48 36

tgatcagttc ttaactcaac caa

<210> 49

<211> 20

<212> DNA

<213> Homo sapiens

<400> 49

ggtctccaat tccatccaga

<210> 50

<211> 20

<212> DNA

<213> Homo sapiens

<400> 50

tggaacatgc ccattaatca

<210> 51

<211> 20

<212> DNA

<213> Homo sapiens

<400> 51

ccatagtgcc gaggtcaagt

<210> 52

<211> 19

<212> DNA

<213> Homo sapiens

<400> 52

aacacctgat gcctggttg

<210> 53

<211> 22 37

<212> DNA

<213> Homo sapiens

<400> 53

gaactgtcta caatggctca cg

<210> 54

<211> 26

<212> DNA

<213> Homo sapiens

<400> 54

tcagcatagt caagaaacca gacaac

<210> 55

<211> 19

<212> DNA

<213> Homo sapiens

<400> 55

tgggctgagg agttcaatc

<210> 56

<211> 23

<212> DNA

<213> Homo sapiens

<400> 56

ggcagtctca tttcctggag atg

<210> 57

<211> 26

<212> DNA

<213> Homo sapiens

<400> 57 38

agtgtctcac ttcaagcacc aagcac

<210> 58

<211> 28

<212> DNA

<213> Homo sapiens

<400> 58

acatttttcc aactgtgaat tttgctgc

<210> 59

<211> 20

<212> DNA

<213> Homo sapiens

<400> 59

aagccatgcc tggttaaact

<210> 60

<211> 21

<212> DNA

<213> Homo sapiens

<400> 60

tgtaaccaaa caccacccat t

<210> 61

<211> 23

<212> DNA

<213> Homo sapiens

<400> 61

tgtcaaaatt taacccacaa tca

<210> 62

<211> 24 39

<212> DNA

<213> Homo sapiens

<400> 62

gcagtctttc accataaggt tagc

<210> 63

<211> 20

<212> DNA

<213> Homo sapiens

<400> 63

atgctgaggagaatttccaa

<210> 64

<211> 27

<212> DNA

<213> Homo sapiens

<400> 64

gctattcatc catctaatct atccatt

<210> 65

<211> 28

<212> DNA

<213> Homo sapiens

<400> 65

cctgccatct ctatttatct tgcatata

<210> 66

<211> 24

<212> DNA

<213> Homo sapiens

<400> 66 40

ataaatggag atagtgggtg gatt

<210> 67

<211> 23

<212> DNA

<213> Homo sapiens

<400> 67

cattaaccat agcatttctt tcc

<210> 68

<211> 22

<212> DNA

<213> Homo sapiens

<400> 68

gaagtagagt ggaatggact gc

<210> 69

<211> 20

<212> DNA

<213> Homo sapiens

<400> 69

gcctgggtga cagagctaga

<210> 70

<211> 21

<212> DNA

<213> Homo sapiens

<400> 70

gccacagtgt gagaagtgtg a 41

<---

Claims (72)

1. A set of synthetic oligonucleotide primers intended for DNA identification of an individual, consisting of pairs of synthetic oligonucleotide primers with nucleotide sequences SNP ID NO: 1-20, characterized in that it additionally contains pairs of synthetic oligonucleotide primers with nucleotide sequences SNP ID NO: 21 -70, while synthetic oligonucleotide primers have the following nucleotide composition: DYS385 (SNP ID NO: 1) F: 5'-TET-AGCATGGGTGACAGAGCTA-3' DYS385 (SNP ID NO: 2) R: 5'-TGGGATGCTAGGTAAAGCTG DYS389 (SNP ID NO: 3) F: 5'-HEX-CCAACTCTCATCTGTATTATCTATG DYS389 (SNP ID NO: 4) R: 5'-TCTTATCTCCACCCACCAGA DYS390 (SNP ID NO: 5) F: 5'-TET-TATATTTTACACATTTTTGGGCC DYS390 (SNP ID NO: 6) R: 5'-TGACAGTAAAATGAACACATTGC DYS391 (SNP ID NO: 7) F: 5'-TET-STATTSATTSAATSATASSASSA DYS391 (SNP ID NO: 8) R: GATTCTTTGTGGTGGGTCTG DYS392 (SNP ID NO: 9) F: 5'-FAM-TCATTAATCTAGCTTTTAAAAACAA DYS392 (SNP ID NO: 10) R: 5'-AGACCCAGTTGATGCAATGT DYS393 (SNP ID NO: 11) F: 5'-FAM-GTGGTCTTCTACTTGTGTCAATAC DYS393 (SNP ID NO: 12) R: 5'-AACTCAAGTCCAAAAATGAGG DYS394 (SNP ID NO: 13) F: 5'-HEX-CTACTGAGTTTCTGTTATAGT DYS394 (SNP ID NO: 14) R: 5'-ATGGCATGTAGTGAGGACA DYS437 (SNP ID NO: 15) F: 5'-FAM-GACTATGGGCGTGAGTGCAT-3' DYS437 (SNP ID NO: 16) R: 5'-GAGACCCTGTCATTCACAGATGA DYS438 (SNP ID NO: 17) F: 5'-FAM-CCAAAATTAGTGGGGAATAGTTG-3' DYS438 (SNP ID NO: 18) R: 5'-GATCACCCAGGGTCTGGAGTT-3' DYS439 (SNP ID NO: 19) F: 5'-TET-TCGAGTTGTTATGGTTTTAGGTCT-3' DYS439 (SNP ID NO: 20) R: 5'-GTGGCTTGGAATTCTTTTACCC-3' DYS442 (SNP ID NO: 21) F: 5'- HEX-CCCCAAGTCCCCAAAGTGTGT-3' DYS442 (SNP ID NO: 22) R: 5'-CACTCATTGA TTGATGGGCGTTT-3' DYS445 (SNP ID NO: 23) F: 5'- FAM-AGTTAAGAGCCCCACCTTCCTG-3' DYS445 (SNP ID NO: 24) R: 5'-TTTTGGTGGCATAATCTCAGCTC-3' DYS448 (SNP ID NO: 25) F: 5'- FAM-TGGGAGAGGCAAGGATCCAA-3' DYS448 (SNP ID NO; 26) R: 5'-CCAGACCGGCCAGAAATATGAC-3' DYS449 (SNP ID NO: 27) F: 5'- FAM-TTTTCCCTTAACTTGTGTGATTTTT-3' DYS449 (SNP ID NO: 28) R: 5'-AGGTTGGACAACAAGAGTAAGACAG-3' DYS456 (SNP ID NO: 29) F: 5'- FAM-GGACCTTGTGATAATGTA-3' DYS456 (SNP ID NO: 30) R: 5'-GTTTTGGGCTGAGTTGATGGG-3' DYS458 (SNP ID NO: 31) F: 5'- FAM-AGCAACAGGAATGAAACTCCAAT-3' DYS458 (SNP ID NO: 32) R: 5'-GGAGGGTGGGCGTGGTGG-3' DYS460 (SNP ID NO: 33) F: 5'- ROX-GAGGAATCTGACACCTCTGACA-3' DYS460 (SNP ID NO: 34) R: 5'-GTCCATATCATCTATCCTCTGCCTA-3' DYS481 (SNP ID NO: 35) F: 5'- JOE-AGGAATGTGGCTAACGCTGT-3' DYS481 (SNP ID NO: 36) R: 5'-ACAGCTCACCAGAAGGTTGC-3' DYS504 (SNP ID NO: 37) F: 5'- HEX-TCTACACCACTGTGCCAAGC-3' DYS504 (SNP ID NO: 38) R: 5'-GGCAACAGAGCAACCCCTCT-3' DYS505 (SNP ID NO: 39) F: 5'- FAM-TCTGGCGAAGTAACCCAAAC-3' DYS505 (SNP ID NO: 40) R: 5'-TCGAGTCAGTTCACCAGAAGG-3' DYS518 (SNP ID NO: 41) F: 5'- JOE-GGCAACACAAGTGAAACTGC-3' DYS518 (SNP ID NO: 42) R: 5'-TCAGCTCTTACCATGGGTGAT-3' DYS525 (SNP ID NO: 43) F: 5'- HEX-ATTCACACCATTGCACTCCA-3' DYS525 (SNP ID NO: 44) R: 5'-GAGGCGGAGCTTTTAGTGAG-3' DYS531 (SNP ID NO: 45) F: 5'- FAM-GACCCACTGGCATTCAAATC-3' DYS531 (SNP ID NO: 46) R: 5'-TGCTCCCTTTCTTTGTAGACG-3' DYS533 (SNP ID NO: 47) F: 5'- JOE-CATCTAACATCTTTGTCATCTACC-3' DYS533 (SNP ID NO: 48) R: 5'-TGATCAGTTCTTAACTCAACCAA-3' DYS537 (SNP ID NO: 49) F: 5'- HEX-GGTCTCCAATTCCATCCAGA-3' DYS537 (SNP ID NO: 50) R: 5'-TGGAACATGCCCATTAATCA-3' DYS552 (SNP ID NO: 51) F: 5'- FAM-CCATAGTGCCGAGGTCAAGT-3' DYS552 (SNP ID NO: 52) R: 5'-AACACCTGATGCCTGGTTG-3' DYS570 (SNP ID NO: 53) F: 5'- ROX-GAACTGTCTACAATGGCTCACG-3' DYS570 (SNP ID NO: 54) R: 5' -TCAGCATAGTCAAGAAACCAGACAAC-3' DYS576 (SNP ID NO: 55) F: 5'- ROX-TGGGCTGAGGAGTTCAATC-3' DYS576 (SNP ID NO: 56) R: 5'-GGCAGTCTCATTTCCTGGAGATG DYS635 (SNP ID NO: 57) F: 5'- FAM-AGTGTCTCACTTCAAGCACCAAGCAC-3' DYS635 (SNP ID NO: 58) R: 5'-ACATTTTTTCCAACTGTGAATTTTGCTGC-3' DYS643 (SNP ID NO: 59) F: 5'- FAM-AAGCCATGCCTGGTTAAACT-3' DYS643 (SNP ID NO: 60) R: 5'-TGTAACCAAACACCACCCATT-3' YCAII (SNP ID NO: 61) F: 5'- HEX-TGTCAAAATTTAACCCACAATCA-3' YCAII (SNP ID NO: 62) R: 5'-GCAGTCTTTCACCATAAGGTTAGC-3' GATA H4.1 (SNP ID NO: 63) F: 5'- HEX-ATGCTGAGGAGAATTTCCAA-3' GATA H4.1 (SNP ID NO: 64) R: 5'-GCTATTCATCCATCTAATCTATCCATT-3' Y-GATA-A10 (SNP ID NO: 65) F: 5'- NED-CCTGCCATCTCTATTTATCTTGCATATA-3' Y-GATA-A10 (SNP ID NO: 66) R: 5'-ATAAATGGAGATAGTGGGTGGATT-3' GGAAT1B07 (SNP ID NO: 67) F: 5'- NED-CATTAACCATAGCATTTCTTTCC-3' GGAAT1B07 (SNP ID NO: 68) R: 5'-GAAGTAGAGTGGAATGGACTGC-3' DYF387S1 (SNP ID NO: 69) F: 5'- FAM-GCCTGGGTGACAGAGCTAGA-3' DYF387S1 (SNP ID NO: 70) R: 5'-GCCACAGTGTGAGAAGTGTGA-3'. 2. A method for identifying genotypes for personal identification using a system of microsatellite DNA markers of the Y chromosome, characterized in that they use a set of primers according to claim 1, while genotyping 38 short tandem repeats localized on the non-recombining region of the Y chromosome (YSTR) is carried out. , on a genetic DNA analyzer using capillary gel electrophoresis, using the PCR method and fragment analysis of fluorescently labeled DNA amplicons, use male DNA samples isolated from any biological material in accordance with standard methods that ensure high-quality purification of DNA from proteins and impurities and preventing the degradation of the material during the isolation, when performing capillary electrophoresis, fragments synthesized during PCR, having a fluorescent TEX tag (4,7,2',7'-tetrachloro-6-carboxyfluorescein) in the length range 360-420 bp. , correspond to alleles 9-23 tandem repeats in the DYS385 locus, fragments having a TET fluorescent mark in the length range of 196-224 bp, correspond to alleles 19-26 tandem repeats in the DYS390 locus, fragments having a TET fluorescent mark in the length range 277 -289 bp, correspond to alleles 9-12 tandem repeats in the DYS391 locus, fragments having a fluorescent TET tag in the length range 232-260 bp, correspond to alleles 8-15 tandem repeats in the DYS439 locus, fragments having a fluorescent FAM tag (6-carboxyfluorescesin) in the length range 243-259 bp, correspond to alleles of 10-15 tandem repeats in the DYS392 locus, fragments having a fluorescent FAM tag in the length range 116-128 bp, correspond to alleles 12- 15 tandem repeats in the DYS393 locus, fragments with a fluorescent FAM mark in the length range of 174-190 bp, correspond to alleles of 6-10 tandem repeats in the DYS437 locus, fragments with a FAM fluorescent mark in the length range of 311-326 bp ., correspond to alleles 9-12 tandem repeats in the DYS438 locus, fragments having a fluorescent FAM mark in the length range of 251-263 bp, correspond to alleles 9-12 tandem repeats in the DYS445 locus, fragments having a fluorescent FAM mark in the length range 307-343 bp, correspond to alleles 18-24 tandem repeats in the DYS448 locus, fragments having a fluorescent FAM label in the length range 264-313 bp, correspond to alleles 25-37 tandem repeats in the DYS449 locus, fragments having fluorescent FAM mark in the length range of 141-157 bp, correspond to alleles of 17-17 tandem repeats in the DYS456 locus, fragments having a fluorescent FAM mark in the length range of 106-156 bp, correspond to alleles of 12-24 tandem repeats in locus DYS458, fragments having a fluorescent FAM mark in the length range 165-181 bp correspond to alleles of 11-15 tandem repeats in the DYS505 locus, fragments having a fluorescent FAM mark in the length range 102-127 bp correspond to alleles 8-14 tandem repeats in the DYS531 locus, fragments with a fluorescent FAM tag in the length range of 236-256 bp correspond to alleles 7-12 tandem repeats in the DYS552 locus. fragments having a fluorescent FAM mark in the length range 238-274 bp correspond to alleles 17-26 tandem repeats in the DYS635 locus, fragments having a fluorescent FAM mark in the length range 123-158 bp correspond to alleles 7-14 tandem repeats in the DYS643 locus. fragments with a fluorescent FAM tag in the length range of 240-272 bp correspond to alleles of 27-35 tandem repeats in the DYF387 locus, fragments with a fluorescent HEX tag (4,7,2',4',5',7' -hexachloro-6-carboxyfluorescein) in the length range 190-206 bp, correspond to alleles 13-17 tandem repeats in the DYS394 locus, fragments having a fluorescent HEX tag in the length range 242-262 bp, correspond to alleles 7- 12 tandem repeats at the DYS389I locus. fragments having a fluorescent HEX tag in the length range of 360-382 bp correspond to the DYS389II locus, alleles of the DYS389II locus are determined by subtracting the length of the DYS389I fragment from the length of the fragment of the range 360-382, the resulting range is 110-134 bp. corresponds to 14-20 tandem repeats, fragments having a HEX fluorescent tag in the length range of 298-322 bp, corresponds to alleles of 10-16 tandem repeats in the DYS442 locus, fragments having a HEX fluorescent tag in the length range 265-296 bp ., correspond to alleles of 12-20 tandem repeats in the DYS504 locus, fragments having a HEX fluorescent mark in the length range of 305-321 bp, correspond to alleles of 9-13 tandem repeats in the DYS525 locus, fragments having a HEX fluorescent mark in the length range 155-179 bp, correspond to alleles 7-13 tandem repeats in the DYS537 locus, fragments having a fluorescent HEX tag in the length range 147-165 bp, correspond to alleles 17-26 tandem repeats in the YCAII locus, fragments having fluorescent HEX tag in the length range of 134-152 bp, correspond to alleles of 9-14 tandem repeats in the Y-GATA-H4.1 locus, fragments having a fluorescent ROX (carboxy-X-rhodamine) tag in the length range of 102-122 bp, correspond to alleles 8-13 tandem repeats in the DYS460 locus, fragments having a fluorescent ROX tag in the length range 242-282 bp, corresponding to alleles 13-23 tandem repeats in the DYS570 locus, fragments having a fluorescent ROX tag in the length range 168-204 bp, correspond to alleles 13-22 tandem repeats in the DYS576 locus, fragments with a JOE fluorescent label in the length range 117-157 bp, correspond to alleles 189-32 tandem repeats in the DYS481 locus, fragments with a fluorescent label JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein) in the length range of 252-296 bp, correspond to alleles of 25-36 tandem repeats in the DYS518 locus, fragments , having a JOE fluorescent tag in the length range of 200-224 bp, correspond to alleles of 9-15 tandem repeats in the DYS533 locus; fragments bearing a fluorescent NED tag correspond to the Y-GATA-A10 or GGAAT1B07 locus.